ABSTRACT
For studying nucleo-cytoplasmic relations during development various selective influences on the nucleus and cytoplasm are widely used as the main method of experimental analysis. However, the application of such techniques presents difficulties both in obtaining evidence that shows the specificity of a nuclear or cytoplasmic effect by a chosen agent and in the quantitative evaluation of the extent of damage.
In this paper a method is described for differentiating between nuclear and cytoplasmic sites of action of a given agent as well as for evaluating quantitatively the extent of nuclear damage. The method is based on the determination of the morphogenetic activity of nuclei at different stages of embryonic development. As has been previously shown, after complete inactivation of nuclei (for instance, by heavy doses of radiation) development proceeds up to the stages programmed for by the genetic cell apparatus (Neyfakh, 1959, 1964), Within a certain range of doses ionizing radiation may be regarded as a factor selectively affecting the nucleus, which is proved for instance by the identity of androgenetic and gynogenetic embryos obtained after heavy irradiation of ova and spermatozoa.
On comparing the ability to develop of embryos with nuclei inactivated at different developmental stages, one can determine both the moment of onset of morphogenetic nuclear function and its intensity (Fig. 1). Thus in loach embryos morphogenetic nuclear function does not begin until practically the mid-blastula stage (6 h at 21 °C). Development from fertilization to the late blastula stage (9 h) proceeds utilising genetic information obtained during oogenesis. This is concluded from the cessation of development of embryos exposed to heavy doses of radiation at different developmental stages up to 6 h. The arrest takes place at the 9h stage and doesn’t depend on the time of irradiation. When embryos are irradiated at the stages from 6 to 8 – 9 h, the arrest of development does depend on the time of irradiation, and embryos irradiated at the late blastula stage are able to proceed through the whole of gastrulation (9 – 18 h). Thus the morphogenetic nuclear function appearing during the 2 to 3 h from the 6 h stage to the 8 – 9 h stage provides the information for the whole process of gastrulation (from the 9 h stage to the 18 h stage). The intensity of morphogenetic function may be expressed as the number of hours of development potentially programmed for by nuclei during 1 h of their previous functioning. On the graph it may be determined by the slope of the curve, or more precisely, by the tangent of slope angle. Similar data, but somewhat shifted in time, are obtained when the time (in hours) from the moment of irradiation to the moment of death of a proportion (for instance, 80 %) of embryos is used as a criterion. Unlike the stage of arrest, the time of 80 % mortality of embryos may be deter-mined with accuracy. This criterion has been used in our investigation. The activity of nuclei allowing the survival of embryos is denoted conventionally as ‘morphogenetic function’, although in this case the term is not quite precise.
Dependence of the time of arrest in the course of development (ordinate) on the time at inactivation of nuclei (abscissa), α—the angle of the slope of the curve. M.N.F.—morphogenetic nuclear function.
One may expect that a partial inhibition of the genetic apparatus will reduce the intensity of later morphogenetic activity and will manifest itself in a decrease in life span of embryos with nuclei inactivated during the period of morphogenetic activity.
The percentage survival after irradiation seems to be determined not by the whole genome but only by a limited number of genes. However, this makes no difference, as in such a case a decrease in embryonic life span indicates that a given agent affects the nucleus. Damaging agents which do not affect the genetic apparatus may induce a decrease in the life span of embryos, but this decrease does not depend on the developmental stage at which inactivation occurred. This difference enables us to differentiate between nuclear and cytoplasmic damage.
In this work the following specific agents have been used to influence the nuclear apparatus: (1) moderate doses of ionizing radiation; (2) treatment with actinomycin; (3) elimination of one of the chromosome sets (haploid development) or its replacement by another set of chromosomes (hybridization). Sodium desoxycholate and sodium dodecylsulphate solutions, which damage lipoprotein cell membranes, served as factors assumed to produce a cytoplasmic effect.
MATERIALS AND METHODS
Experiments were carried out on embryos of the loach Misgurnus fossilis L. Developmental stages were expressed as hours of normal development at 21 °C.
At early developmental stages embryos were subjected to the action of one of the above agents. The dose was chosen so as to permit the survival of embryos up to the end of gastrulation. Embryos were then X-irradiated with heavy doses (10–35 kr, 15 mA, 190 kV without filter) at successive developmental stages at 10–60 min intervals. As has been previously shown, such doses result in a complete inactivation of the nucleus, the effect being constant within a given dose range (Neyfakh, 1959). 200–400 eggs were taken at each time of irradiation. To determine the time of 80 % mortality, dead embryos were counted each hour. Appropriate curves were constructed, the percentage of dead embryos plotted against time. The time of 80 % mortality of embryos could be calculated precisely (± 10–15 min) from these curves. The results obtained were plotted on a graph showing the dependence of time of 80 % mortality of embryos on the time of irradiation. The slope of the curve reflects the intensity of morphogenetic nuclear function.
RESULTS
1. Haploid embryos
Gynogenetic haploid embryos were obtained by irradiating spermatozoa prior to fertilization with heavy doses (about 60–70 kr), which caused a 100 % haploidy of embryos (Bakulina, Pokrovskaga & Romashov, 1962). As to the rate and character of development, such embryos do not differ from diploid ones at early developmental stages, their defects being revealed only after gastrulation. Almost all of them reach the stage of hatching.
Fig. 2a shows the changes in life span of haploid and diploid embryos after irradiation at different developmental stages. One can see that both in diploid and haploid embryos nuclear function, which ensures later viability of embryos, appears approximately from the 4 h stage (early blastula) to the 8–9 h stage (late blastula).
Morphogenetic nuclear function in diploid (2n) and haploid (n) embryos.
(a) Dependence of the time of 80 % mortality (ordinate) on the time at radiation inactivation of nuclei (abscissa). Dose—10 kr.
(b) Dependence of intensity of morphogenetic nuclear function (ordinate) on the developmental stage (abscissa). The graph was drawn using the data represented in Fig. 2a. The intensity of nuclear morphogenetic function was determined by the increase in the embryonic life span when irradiation was carried out an hour later. Thearea(S) contained by this curve gives the summation of activity of nuclei in early development.
Morphogenetic nuclear function in diploid (2n) and haploid (n) embryos.
(a) Dependence of the time of 80 % mortality (ordinate) on the time at radiation inactivation of nuclei (abscissa). Dose—10 kr.
(b) Dependence of intensity of morphogenetic nuclear function (ordinate) on the developmental stage (abscissa). The graph was drawn using the data represented in Fig. 2a. The intensity of nuclear morphogenetic function was determined by the increase in the embryonic life span when irradiation was carried out an hour later. Thearea(S) contained by this curve gives the summation of activity of nuclei in early development.
Although both curves presented in Fig. 2a are very similar, it is clear that haploid embryos die much earlier than diploid ones when irradiated at the 5–9 h stages. When nuclei are inactivated at the 9–10 hr stage the curves coincide. Interesting results may be obtained when the intensity of nuclear function in haploid and diploid embryos is compared (Fig. 2 b). The total values of nuclear activity during the period of their functioning in haploid and diploid embryos appear to be approximately the same, indicating that they have the same total amount of nuclear ‘production’. However, in haploids the bulk of this ‘production’ is elaborated later than in diploids. Compared to diploid nuclei the less intense activity of haploids at early developmental stages is compensated by their more intense activity at later stages; the peak of the curve showing the intensity of nuclear function in haploids is shifted to the right in Fig. 2b. One can suggest that this late compensation is not able to normalize completely the development of embryos disturbed by the deficiency of nuclear function in the initial period. This appears to be one of the likely causes of the haploid syndrome.
2. Hybrid embryos
If loach eggs are fertilized by goldfish sperm, fertilization proceeds normally, and development does not differ from that of normal diploid loach embryos. However, before hatching embryos acquire such features of the goldfish as early eye pigmentation and typical yolk form. After hatching, the development of hybrid larvae is delayed and they gradually die. It has been shown (Neyfakh & Radzievskaya, 1967) that in hybrids a major part (up to ) of the paternal chromosome set is eliminated. Thus it might be expected that the paternal genotype would show a certain morphogenetic activity, but it should be less than that of the maternal genotype due to the elimination of a portion of chromosomes and/or incompatibility of paternal chromosomes with maternal cytoplasm. Consequently the intensity of nuclear function in hybrid embryos should be higher than in haploid loach embryos but less than in diploid ones. In Fig. 3 it is seen that the survival curve for hybrid embryos after radiation inactivation of nuclei lies between the corresponding curves for haploid and diploid loach embryos. Of particular interest is the fact that at the late blastula stage, when the intensity of morphogenetic function in loach embryos is temporarily declining, no decrease is observed in hybrid embryos. These experiments show that hybrids follow the paternal species in this respect. This suggests that the time pattern of morphogenetic nuclear function is under genetic control.
3. Irradiation with moderate doses of ionizing radiation
Diploid embryos were irradiated with doses from 500 to 2000 r at the 1–5 h developmental stages. The irradiation didn’t markedly affect early developmental stages, although it did cause mortality of some embryos at later stages. After this preliminary irradiation, nuclei were completely inactivated by 35 kr irradiation at different times of development. Irradiation is known to produce the same effect within the dose range from 10 to 40 kr. Thus it appears that summation of the dose of preliminary irradiation with that of inactivation is unlikely to affect the survival of embryos.
Fig. 4 shows the changes in life span of normal and early irradiated embryos, with nuclei inactivated at different later developmental stages. For embryos irradiated with 750 r the life expectancy is decreased, and the summed intensity of nuclear function is also reduced. Thus, irradiation-induced partial loss of genetic material causes a decrease in intensity of morphogenetic nuclear function at early developmental stages as well as anomalies of development at later times.
4. Action of actinomycin
In this work actinomycin prepared in Moscow University (cbrysomaline) was used. Actinomycin hardly penetrates fresh-water fish embryos. On intact loach embryos a marked effect of actinomycin treatment can be obtained only if the latter is applied during the first moments after fertilization in as high concentrations as 100–200 μg/ml. The development of isolated blastoderms is blocked at concentrations 10–20μg/ml. It means that cells show their usual sensitivity to actinomycin, but only a small amount can penetrate the embryo from the medium.
In our experiments fertilized eggs were placed in aqueous actinomycin solution for 1 h and then washed. The following concentrations were used: 25, 50, 100 and 200 μg/ml. Development proceeded quite normally to the late blastula stage. Further development at a concentration of 200 μg/ml was completely arrested. Treatment with 100 μg/ml resulted in a partial arrest at the same developmental stage: a proportion of embryos proceeded through the first steps of gastrulation. After treatment with 50μg/ml, embryos gastrulated and developed further, although some abnormalities and delay were observed in their develop-ment. After the action of 25μg/ml the first half of embryonic development proceeded normally.
In Fig. 5 the data are summarized on the life span of normal and actinomycintreated embryos after nuclear inactivation with 15 kr at different developmental stages. One can see that actinomycin, at all concentrations used, markedly diminished the life span of embryos. This was the consequence of a decrease in the intensity of nuclear function on the whole proportional to the antibiotic concentration. Hence actinomycin even at concentrations which don’t induce morphological changes affects the intensity of nuclear function.
Morphogenetic nuclear function in normal (• — —•) and actinomycin pre-treated embryos. Actinomycin concentrations, 25 (○ — ○), 50 (× — ×), 100 (○ ○) and 200 (○ • ○ •) μg/ml. Dose, 15 kr.
5. Sodium desoxycholate and sodium dodecylsulphate action
Embryos were treated with sodium dodecylsulphate solution (SDS) (25 μg/ml) for 1 h and morphogenetic nuclear function was investigated thereafter. In another experiment embryos were placed in SDS (5 μg/ml) after radiation inactivation of nuclei and kept there until death. In non-irradiated control embryos, treatment with these concentrations didn’t cause death up to the end of gastrulation. Fig. 6 shows that treatment with SDS accelerated death of irradiated embryos. However, unlike the action of the nuclear factors referred to above, SDS accelerated the death of embryos with inactivated nuclei whether inactivation occurred during the period of morphogenetic function or before it.
Morphogenetic nuclear function in normal (• — •) and sodium dodecylsulphate (SDS) treated embryos. Embryos were treated for 1 h with 25 /‘g/ml SDS before radiation inactivation of nuclei (○ — — ○) or with 5 μg/ml after irradiation (× – – × ). Dose, 30 kr.
The angle of the slope of the survival curve practically didn’t change during the period of morphogenetic activity. In other words, SDS didn’t change intensity of morphogenetic nuclear function, but only accelerated the realization of nuclear, irradiation-induced, damage. It is of interest that SDS produced the same effect when applied after radiation inactivation of nuclei, while a nuclear agent applied after radiation inactivation of nuclei could not be expected to produce any effect.
Treatment with sodium desoxycholate (DOC) at a concentration 0·75 mg/ml for 1 h prior to irradiation and with 0·5 mg/ml (continuously) after irradiation affected nuclei in a similar way to that observed for SDS. In this case also the cytoplasmic character of action of this agent is revealed by the fact that it accelerates embryonic death independent of the moment of irradiation, and remains effective when appplied after radiation inactivation of nuclei (Fig. 7).
Morphogenetic nuclear function in normal (• — •) and desoxycholate (DOC) treated embryos. Embryos were treated for 1 h with 0· 75 mg/ml desoxycholate (○ — — ○) before radiation inactivation of nuclei or with 0· 5 mg/ml after irradiation (× — — ×). Dose, 30 kr.
DISCUSSION
All the factors investigated which affect the genetic apparatus of the cell (haploidy, hybridization, ionizing radiation and actinomycin treatment) signifi-cantly influence the character and intensity of morphogenetic nuclear function in early development. The method described in this paper allows the detection of the effect of these factors at stages and doses which do not reveal this action morphologically.
Agents affecting cytoplasmic cell structures (sodium dodecylsulphate and desoxycholate) do not influence morphogenetic nuclear function. They accelerate embryonic death to the same extent independent of the moment of inactivation of the genetic apparatus of the cell, either during the period of morphogenetic nuclear activity or before it. This would enable us to differentiate nuclear and cytoplasmic action by damaging agents of an unknown nature and to evaluate quantitatively the extent of nuclear damage.
It seems of interest to compare the data described in the present paper with such direct measures of nuclear activity as the intensity of RNA synthesis. In haploid loach embryos nuclear RNA synthesis proceeds twice as slowly at the mid-blastula stage (6–8 h) as similar synthesis in diploid embryos. However, by the mid-gastrula stage (10 h) the intensity of RNA synthesis in diploid and haploid embryos becomes practically the same (Timofeeva, Neyfakh & Kafiani, 1967). Similar results have been obtained in our experiments. At very early stages the morphogenetic nuclear activity in haploids is sharply decreased as compared with that of diploids, but at subsequent developmental stages (8-10 h) the decrease has been compensated. As a result, the total amount of nuclear function appears to be approximately the same in haploids and diploids. In haploid embryos the number of nuclei at this time is approximately 1-5 times higher than in diploids (Rott & Sheveleva, 1968). Hence the total amount of genetic material in haploids is less than in diploids. One can suggest that each chromosome of a haploid cell is capable of intensifying its function by revealing higher genetic activity and/or synthesizing more RNA than a homologous, chromosome of the diploid cell. Such compensation of gene dosage has been found for the X-chromosome of Drosophila males for both genetic activity (Müller, League & Offermann, 1932) and RNA synthesis (Mukherjee & Beermann, 1965).
Actinomycin treatment (100 μg/ml) causes approximately a 50 % decrease in RNA synthesis (Timofeeva, personal communication). According to our data, the same treatment decreases nuclear function to about half intensity. One would not expect a complete coincidence of the data on intensity of RNA synthesis and nuclear function determined by the method described, but the preceding examples show that phenomena of the same type underlie both inhibitions.
Thus the use of the method described for comparing haploid and diploid embryos permits clarification of the mechanism of compensation of developmental processes at the change of ploidy and understanding of the causes of the haploid syndrome. The determination of changes in the intensity of nuclear function as an index of nuclear damage may be used to solve certain radiobiological problems as well. The high sensitivity of the method as well as its quantitative character suggests that it can be used to detect the action of various chemical and physical agents on the cell nucleus.
SUMMARY
A method is suggested for distinguishing nuclear and cytoplasmic action by various harmful agents permitting quantitative evaluation of the extent of nuclear damage. The method is based on the determination of morphogenetic nuclear activity at different developmental stages.
The elimination of or damage to some portion of the genetic material (haploidy, distant hybridization, irradiation and actinomycin treatment) results in a decrease of nuclear activity in the period of morphogenetic function. In haploid embryos the absence of one of the chromosome sets may be partially compensated by intensified functioning of a single chromosome set.
Agents affecting cytoplasmic cell structures (sodium dodecylsulphate, sodium desoxycholate) do not influence directly morphogenetic nuclear function, but cause a decrease of embryonic life span independent of the time of nuclear inactivation.
There exists a certain quantitative correlation between morphogenetic nuclear activity and RNA synthesis.